Norihiko Nakano

Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat.

Bone marrow stromal cells (BMSCs) have been studied as effective transplants for the treatment of spinal cord injury (SCI). Our previous study showed that BMSCs infused into the cerebrospinal fluid (CSF) exhibited distinct effects on the recovery of acute SCI. The present study examined the effects of BMSCs in sub-acute SCI (2weeks post-injury) by transplanting them directly into the lesion. The spinal cord was crush-injured at the Th8-9 level in rats, and 2weeks later, cultured BMSCs (5x10(5)) derived from GFP-transgenic rats of the same strain were transplanted into the lesion. Tissue repair and nerve regeneration were examined by immunohistochemistry and electron microscopy. GFP-labeled BMSCs survived as cell assemblies in the spinal cord for 1-2weeks after transplantation. The dorsal side of BMSC assemblies in the spinal cord usually showed an expanded GFAP-negative, astrocyte-devoid area, in which extracellular matrices including collagen fibrils were deposited. Numerous regenerating axons associated with Schwann cells grew out through such astrocyte-devoid extracellular matrices. Ascending (CGRP-containing) and descending (5HT- and TH-containing) axons were included in these regenerating axons. Regenerated axons were myelinated by Schwann cells beyond 2weeks post-transplantation. Cavity formation was reduced in the cell transplantation group. Locomotory behavior assessed by the BBB scale improved to 9.8 points in the cell transplantation group, while it was to 5.5-5.7 in the control. BMSC transplantation into lesions of advanced SCI has markedly beneficial effects on tissue repair and axonal outgrowth, leading to improved locomotion in rats.

A Secreted Type of β1,6-N-Acetylglucosaminyltransferase V (GnT-V) Induces Tumor Angiogenesis without Mediation of Glycosylation A NOVEL FUNCTION OF GnT-V DISTINCT FROM THE ORIGINAL GLYCOSYLTRANSFERASE ACTIVITY

Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that β1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltransferase. A secreted type of GnT-V protein itself promoted angiogenesis in vitro and in vivo at physiological concentrations. The highly basic domain of GnT-V induced the release of fibroblast growth factor-2 from heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, leading to angiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the inhibition of tumor angiogenesis.

Antioxidative Effects of a New Lychee Fruit-Derived Polyphenol Mixture, Oligonol, Converted into a Low-Molecular Form in Adipocytes

In this study we investigated the antioxidative effects of Oligonol (Amino Up Chemical Co., Ltd., Sapporo, Japan), a new polyphenol, in adipocytes. The levels of reactive oxygen species (ROS) and the expression of adipokine genes decreased in HW mouse white adipocytes upon treatment with Oligonol as compared to control cells. The transcriptional activity of nuclear factor-kappaB (NF-κB) and the activation of extracellular signal-regulated kinase (ERK) 1/2 were also down-regulated by Oligonol. In addition, when C57BL/6J mice were fed a high fat diet (HFD) for 5 weeks, the levels of epididymal white adipose tissue (WAT) mass and lipid peroxidation in WAT both increased, but Oligonol intake clearly inhibited such HFD-induced increases. Furthermore, dysregulated expression of genes for adipokines in WAT of mice fed solely a HFD was attenuated by Oligonol intake. These results suggest that Oligonol has antioxidative effects and that it attenuates HFD-induced dysregulated expression of genes for adipokines in adipocytes.

Effects of Bone Marrow Stromal Cell Transplantation through CSF on the Subacute and Chronic Spinal Cord Injury in Rats

It has been demonstrated that the infusion of bone marrow stromal cells (BMSCs) through the cerebrospinal fluid (CSF) has beneficial effects on acute spinal cord injury (SCI) in rats. The present study examined whether BMSC infusion into the CSF is effective for subacute (1- and 2-week post-injury), and/or chronic (4-week post-injury) SCI in rats. The spinal cord was contused by dropping a weight at the thoracic 8-9 levels. BMSCs cultured from GFP-transgenic rats of the same strain were injected three times (once weekly) into the CSF through the fourth ventricle, beginning at 1, 2 and 4 weeks post-injury. At 4 weeks after initial injection, the average BBB score for locomotor assessment increased from 1.0–3.5 points before injection to 9.0-10.9 points in the BMSC-injection subgroups, while, in the PBS (vehicle)-injection subgroups, it increased only from 0.5–4.0 points before injection to 3.0-5.1 points. Numerous axons associated with Schwann cells extended longitudinally through the connective tissue matrices in the astrocyte-devoid lesion without being blocked at either the rostral or the caudal borders in the BMSC-injection subgroups. A small number of BMSCs were found to survive within the spinal cord lesion in SCI of the 1-week post-injury at 2 days of injection, but none at 7 days. No BMSCs were found in the spinal cord lesion at 2 days or at 7 days in the SCI of the 2-week and the 4-week post-injury groups. In an in vitro experiment, BMSC-injected CSF promoted the survival and the neurite extension of cultured neurons more effectively than did the PBS-injected CSF. These results indicate that BMSCs had beneficial effects on locomotor improvement as well as on axonal regeneration in both subacute and chronic SCI rats, and the results also suggest that BMSCs might function as neurotrophic sources via the CSF.

Bone marrow-derived mononuclear cell transplantation in spinal cord injury patients by lumbar puncture.

PURPOSE This study was conducted to assess the safety and feasibility of intrathecal transplantation of autologous bone marrow-derived mononuclear cells for the treatment of patients with spinal cord injury. METHODS Ten patients were included in the study. Approximately 120 ml of bone marrow aspirate was obtained from bilateral iliac bone of patients with spinal cord injury. Isolation of mononuclear cells was performed using Ficoll density-gradient centrifugation. Bone marrow mononuclear cells were transplanted into cerebrospinal fluid by lumbar puncture. Functional tests were performed prior to the cell transplantation and six months after cell transplantation. The patients were carefully observed for up to six months. RESULTS In 5 patients with AIS A prior to cell transplantation, 1 patient converted to AIS B six months after cell transplantation. In 5 patients with AIS B, 1 patient converted to AIS D and 2 patients to AIS C. MRI did not show any complication. Two patients showed slight anemia after aspiration of bone-marrow cells, which returned to normal level within a several weeks. CONCLUSION The results of this study suggest that this method may be safe and feasible.

Transplantation of choroid plexus epithelial cells into contusion-injured spinal cord of rats.

Purpose: The effect of the transplantation of choroid plexus epithelial cells (CPECs) on locomotor improvement and tissue repair including axonal extension in spinal cord lesions was examined in rats with spinal cord injury (SCI). Methods: CPECs were cultured from the choroid plexus of green fluorescent protein (GFP)-transgenic rats, and transplanted directly into the contusion-injured spinal cord lesions of rats of the same strain. Locomotor behaviors were evaluated based on BBB scores every week after transplantation until 4 weeks after transplantation. Histological and immunohistochemical examinations were performed at 2 days, and every week until 5 weeks after transplantation. Results: Locomotor behaviors evaluated by the BBB score were significantly improved in cell-transplanted rats. Numerous axons grew, with occasional interactions with CPECs, through the astrocyte-devoid areas. These axons exhibited structural characteristics of peripheral nerves. GAP-43-positive axons were found at the border of the lesion 2 days after transplantation. Cavity formation was more reduced in cell-transplanted than control spinal cords. CPECs were found within the spinal cord lesion, and sometimes in association with astrocytes at the border of the lesion until 2 weeks after transplantation. Conclusion: The transplantation of CPECs enhanced locomotor improvement and tissue recovery, including axonal regeneration, in rats with SCI.

Bone marrow stromall cell transplantation for treatment of sub-acute spinal cord injury in the rat

The stigmoid body (STB) is the neurocytoplasmic inclusion abundantly and specifically distributed in the limbic and hypothalamic regions of the normal brain. The STB has been also determined to contain huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, and to be induced by the transfection of HAP1-cDNA into cultured cells (STB/HAP1). Although STB/HAP1 has been reported to have protective functions in Huntington?s disease and spinal-and-bulbar-muscular-atrophy by sequestering the causative proteins, huntingtin and androgen receptor (AR), its physiological functions remain unclear. In the present study, using immunocytochemistry, immunoprecipitation and time-lapse image analysis, we examined subcellular interaction of STB/HAP1 with the steroid-hormone receptors, including AR, estrogen receptors (ER and ER ), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in COS-7 cells cotransfected with HAP1 and each receptor. The results clearly showed that STB/HAP1 sequesters GR and AR via a ligand-binding domain and that the receptors maintain nucleartranslocation activity in response to their specific ligands. When the cells were treated with MG132, a proteasome inhibitor, GR and AR localized outside of STB/HAP1 were clearly translocated into the nucleus as seen in the cells without MG132, while the receptors localized in the STB/HAP1 persisted in their association even after the treatment of their specific ligands. These results suggest that STB/HAP1 has a relaying function to modify the nuclear translocation of GR and AR, which is closely related to the proteasome activity.

NTAK/neuregulin-2 secreted by astrocytes promotes survival and neurite outgrowth of neurons via ErbB3.

NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2 (NRG2), is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4, and transactivates ErbB2. NTAK/NRG2 is structurally homologous to NRG1. The biological function of NTAK/NRG2 still remains unknown, especially in the nervous system, whereas NRG1 is known to be essential for nervous system function. In the present study, we examined the functions of NTAK/NRG2 secreted from astrocytes to neurons. NTAK/NRG2 was expressed in both neurons and astrocytes, as evidenced by immunohistochemical staining and RT-PCR methods. The conditioned medium (CM) from astrocytes promoted survival and neurite outgrowth of neurons. The CM stimulated phosphorylation of ErbB3 in neurons. When phosphorylation of ErbB3 was blocked by AZD8931, an ErbB3 inhibitor, neuronal survival and neurite outgrowth were reduced. Conversely, canertinib, an ErbB4 inhibitor, did not affect survival or neurite outgrowth of neurons. Survival and neurite outgrowth of neurons were lower in CM of NTAK/NRG2-knockdown astrocytes than in the CM of control astrocytes, whereas the CM of NRG1-knockdown astrocytes had little effect on survival and neurite outgrowth. The present study demonstrated that NTAK/NRG2 secreted from astrocytes bound to ErbB3 on neurons, and promoted neuronal survival and neurite extension in vitro.

Analysis of bone marrow stromal cell transplantation for spinal cord injury in the rat

After traumatic damage of the central nervous system (CNS), meningeal fibroblasts receive transforming growth factor(TGF) signal, invade in the lesion site, actively proliferate, and secrete extracellular matrix proteins (Komuta et al., Cell Mol. Neurobiol. 30: 101–111, 2010). Those cells and proteins consistently form a fibrotic scar which is considered as a major obstacle for axonal regeneration after injury. We have previously examined the development of fibrotic scar formation in postnatal mice with a surgical transection of the nigrostriatal dopaminergic pathway. The fibrotic scar was not formed and axonal regeneration occurred in the brain injured at postnatal day 7 (P7), while the fibrotic scar was formed and axons did not regenerate in the brains injured after P14 (Kawano et al., J. Neurosci. Res. 80: 191–202, 2005). Recently, we have demonstrated that cultured meningeal fibroblasts form fibrotic scar-like clusters by an addition of TGF1 (KimuraKuroda et al., Mol. Cell. Neurosci. 43: 177–187, 2010). Using this in vitro model, we examined the relationship between the postnatal development of fibrotic scar formation and the cell responses for TGF1. Meningeal fibroblasts from P0 and P14 rats were isolated, cultured and added with TGF1 (1–10 ng/ml). Meningeal fibroblasts taken from P14 rats responded to TGF1 to actively proliferate, form clusters and express the markers of fibrotic scar fibronectin and type IV collagen. In contrast, meningeal fibroblasts taken from P0 rats less proliferated and did not form clusters. Furthermore, most of fibrotic scar markers were not up-regulated in those cells. The present results agree well with our previous in vivo finding, and further suggest that failure of neonatal meningeal fibroblasts to respond to TGF1 inhibits the fibrotic scar formation and allow the axonal regeneration after brain injury.